Qiaquick pcr purification kit

Gene expression profile of biomarkers were determined by relative target gene quantities from threshold cycles (C_t) normalised against the housekeeping gene GAPDH in FLO-1and BO cell lines through the mRNA extraction using the RNA easy kit (Qiagen, Crawley, UK), cDNA synthesis using the Quick reverse transcription system (Promega) and quantification by real-time PCR (qPCR) with primer sets after stimulating by C. concisus as described previously by Mozaffari Namin et al. (16 (link)). Briefly, products of correct size and sequence were purified using the Qiaquick PCR purification kit and ligated into a vector using the pGEM-T easy vector system I (Promega). Plasmid DNA from candidate clones was isolated and purified using the Wizard plus SV miniprep system (Promega) and the sequence identity verified. Samples were diluted to 10¹⁰ molecules µl^-1, aliquoted and stored at −80°C. qPCR was performed using an iCycler real-time PCR detection system (BioRad laboratories, Hercules, CA, USA) in the presence of iQ SYBR Green Supermix (BioRad). The standard amplification protocol consisted of an initial denaturation step (95°C, 3 minutes), followed by 38 cycles of 95°C for 30 seconds, 30 seconds at appropriate annealing temperature (Table 1) and one final denaturation cycle (95°C, 30 seconds).